Signal Mediated Growth Control by Osmotic Stress

小行星9506987 这项研究解决了细胞如何感知和响应渗透压的基本问题。 在之前由NSF资助的工作中，已经表明酵母酿酒酵母具有称为HOG激酶（由HOG 1编码）和MAP激酶激酶（由PBS 2编码）的生物传感信号转导途径。 缺乏任一基因的突变体在高渗透压诱导的蛋白质表达中具有特定的缺陷，所述蛋白质需要高渗透压调节和高渗透压耐受。 该实验室最近取得了令人兴奋的发现，酵母细胞暴露于渗透压的增加暂时阻止其细胞周期在G2期; hog 1和pbs 2突变体是有缺陷的，这种渗透压应激反应。 这种细胞周期停滞的机制目前尚不清楚，但工作假设是Hog 1 p负调控细胞周期蛋白依赖性蛋白激酶Cdc 28 p的G2特异性功能。 长期目标是从分子水平上了解渗透压是如何调节细胞周期的。 该项目的具体重点是确定HOG通路如何诱导G2期阻滞。 两个主要目标是：（1）研究高渗透压诱导的Hog 1依赖性调节G2期细胞周期蛋白依赖性激酶Cdc 28功能所需的机制;（2）分离和表征HOG途径介导G2期阻滞的分子靶点。 具体实验包括：（1）利用G2期细胞，研究高渗透压和HOG途径突变体对G2期cyclin/Cdc 28复合物的组蛋白H1激酶活性、G2期cyclin/Cdc 28复合物的体内稳定性和G2期cyclin的细胞内含量的影响。 将确定Cks 1或其他已知的Cdc 28相关蛋白是否介导Hog 1对G2特异性Cdc 28功能的调节。还将确定Cdc 28磷酸化的变化是否介导Hog 1诱导的G2期阻滞。 将通过确定渗透胁迫和hog 1突变对细胞周期标志事件的影响来研究Hog 1导致的G2阻滞的生理意义。 2)使用亲和层析和免疫共沉淀分离和表征Hog 1或G2细胞周期蛋白/Cdc 28相关蛋白介导Hog 1诱导的G2停滞。 这一分子分析的结果，细胞周期调控的approximately调节的蛋白激酶级联将提供一个重要的实验范式，在其他真核系统中的类似的渗透反应途径的研究。 这个项目解决了一个信号通路的新特征，这个新特征是由这个研究者在之前的NSF奖中发现的。 信号通路是高渗透胁迫激活基因（HOG），其显然通过细胞分裂影响正常进程。 如果细胞暴露于高渗透条件，例如高盐或糖浓度，它们通常在称为“G2”的时间段内阻止细胞周期，即DNA合成（S期）和有丝分裂（M期）之间的差距。 具有失活HOG 1基因的细胞不会在G2期停止，而是继续分裂，通常是异常的。 从G2到M的转变已被证明部分地由磷酸化酶或激酶的复合物的循环组装控制，磷酸化酶或激酶是Cdc 28（细胞分裂控制28）基因和称为细胞周期蛋白的蛋白质的产物，其在大多数情况下在细胞周期的进展期间循环地产生。在G2期间产生并允许通过G2/M转变的细胞周期蛋白被称为G2细胞周期蛋白。 允许通过G1（有丝分裂和DNA合成之间的差距）/S转换的细胞周期蛋白被称为G1细胞周期蛋白。该项目的重点是G2细胞周期蛋白和Cdc 28激酶形成的复合物的性质如何受到Hog 1的影响。Hog 1的活性、稳定性以及与其他蛋白质的结合都是Hog 1作用的候选者。 为了确定在介导Hog 1诱导的细胞周期停滞的G2细胞周期蛋白/Cdc 28复合物中是否存在其他调节组分，将通过它们与复合物组分的结合来鉴定和分离其他组分。这项工作很重要，因为它可以作为真菌和植物细胞对不断变化的环境的反应的模型，例如部分干燥或盐度增加，这两者都会改变细胞的渗透条件。 ***

9506987 Gustin The research addresses the fundamental problem of how cells sense and respond to osmotic stress. In previous work funded by NSF, it was shown that the yeast Saccharomyces cerevisiae has an osmosensing signal transduction pathway called the HOG kinase (encoded by HOG1) and a MAP kinase kinase (encoded by PBS2). Mutants lacking either gene have specific defects in the high osmolarity-induced expression of proteins need for osmoregulation and osmotolerance. This laboratory recently made the exciting discovery that yeast cells exposed to an increase in osmolarity temporarily arrest their cell cycle in the G2 phase; hog 1 and pbs2 mutants are defective for this osmotic stress response. The mechanism responsible for this cell cycle arrest is currently unknown, but the working hypothesis is that Hog1p negatively regulates a G2-specific function of the cyclin- dependent protein kinase Cdc28p. The long-range goal is a molecular understanding of how the cell cycle is regulated by osmotic stress. The specific focus of this project is to determine how the HOG pathway induces a G2 phase arrest. Two major objectives are (1) to investigate mechanisms required for high osmolarity-induced, Hog1-dependent regulation of the function of the cyclin-dependent kinase Cdc28 in G2, and (2) to isolate and characterize the molecular targets of the HOG pathway that mediate G2 arrest. Specific experiments to meet the stated objectives are: (1) Use G2-phase cells to determine the effect of high osmolarity and HOG pathway mutants on the histone H1 kinase activity of the G2 cyclin/Cdc28 complex, the in vivo stability of the G2 cyclin/Cdc28 complex, and the cellular content of G2 cyclins. It will be determined whether Cks1 or other known Cdc28-associated proteins mediate Hog1 regulation of G2-specific Cdc28 functions. It will also be determined whether changes in Cdc28 phosphorylation mediate Hog1-induced G2 arrest. The physiological significance of G2 arrest by Hog1 will be investigated by determining effects of osmotic stress and the hog1 mutation on cell cycle landmark events. 2) Use affinity chromatography and co-immunoprecipitation to isolate and characterize Hog1- or G2 cyclin/Cdc28-associated proteins mediating Hog1-induced G2 arrest. Results of this molecular analysis of cell cycle regulation by an osmotically-regulated protein kinase cascade will provide an important experimental paradigm for studies on similar osmotic response pathways active in other eukaryotic systems. %%% This project addresses a new feature of a signaling pathway recently discovered in yeast by this investigator in a prior NSF award. The signaling pathway is the High Osmotic-stress activated Genes (HOG) which apparently have influence over the normal progression through cell division. If the cells are exposed to high osmotic conditions, such as high salt or sugar concentrations, they normally arrest the cell cycle in the period of time called "G2", the Gap of time between DNA synthesis (S phase) and mitosis (M phase). Cells with an inactive HOG1 gene, do not arrest in G2, rather they continue on to divide, often abnormally. The transition from G2 into M has been shown to be controlled, in part, by the cyclical assembly of complexes of a phosphorylating enzyme, or kinase, which is the product of the Cdc28 (Cell Division Control 28) gene and proteins called cyclins, which are, in most part, cyclically made during the progression through the cell cycle. The cyclins which are made during G2 and allow progression through the G2/M transition are called the G2 cyclins. Cyclins which allow progression through the G1 (the Gap of time between mitosis and DNA synthesis)/S transistion are called G1 cyclins. The focus of this project is how the nature of the complex formed by G2 cyclins and the Cdc28 kinase is influenced by the Hog1. The activity, stability, and associations with other proteins are all candidates for Hog 1 action. To determine if other regulatory components are present in the G2cyclin/Cdc28 complex which mediate Hog1-induced cell cycle arrest, the other components will be identified and isolated by virtue of their association with components of the complex. This work is important because it may serve as a model for fungal and plant cellular response to a changing environment, such as partial desication or increase in salinity, both of which change the osmotic conditions of the cell. ***